Mutation of foxl2 or cyp19a1a results in female to male sex reversal in XX Nile tilapia

Abstract

It is well accepted that Forkhead box protein L2 (Foxl2) and aromatase (Cyp19a1; the enzyme responsible for estrogen synthesis) are critical for ovarian development in vertebrates. Knockouts of Foxl2 and Cyp19a1 in goat, mouse, and zebrafish have revealed similar but not identical functions across species. Functional analyses of these two genes in other animals are needed to elucidate their conserved roles in vertebrate sexual development. In this study, we established foxl2 and cyp19a1a mutant lines in Nile tilapia. Both foxl2-/- and cyp19a1a-/- XX fish displayed female-to-male sex reversal. Sf1, Dmrt1, and Gsdf were upregulated in the foxl2-/- and the cyp19a1a-/- XX gonads. Downregulation of Cyp19a1a and serum estradiol-17β level, and upregulation of Cyp11b2 and serum 11-ketotestosterone level were observed in foxl2-/- XX fish. The mutant phenotype of foxl2-/- XX individuals could be rescued by 17β-estradiol treatment from 5 to 30 days after hatching (dah). Upregulation of Star1, the enzyme involved in androgen production in tilapia, was also observed in the foxl2-/- XX gonad at 30 and 90 dah. In vitro promoter analyses consistently demonstrated that Foxl2 could suppress the transcription of star1 in a dose-dependent manner. In addition, compared with the control XX gonad, fewer germ cells were detected in the foxl2-/- XX, cyp19a1a-/- XX, and control XY gonads 10 dah. These results demonstrate that Foxl2 promotes ovarian development by upregulating Cyp19a1a expression and repressing male pathway gene expression. These results extend the study of Foxl2 and Cyp19a1a loss of function to a commercially important fish species.

Mutation ofcyp19a1bresults in sterile males due to efferent duct obstruction in Nile tilapia

“…Due to the teleost-specific whole-genome duplication, teleost fish have two genes encoding aromatase, cyp19a1a and cyp19a1b. In tilapia, no Cyp19a1a expression was detected in XY gonads, suggesting that Cyp19a1a plays a dispensable role in male reproduction, which has been confirmed previously by loss-of-function assays (Zhang et al, 2017). Different from zebrafish and spotted scat, in which Cyp19a1b was expressed in all types of germ cells in the testes, as well as Sertoli cells in the latter (Caulier et al, 2015;Chaube et al, 2015;Liu et al, 2015), Cyp19a1b was found to be expressed in the Leydig cells of tilapia testes in the present study, similar to the cellular location of Cyp19a1 in mammalian testes (Bilińska et al, 2000;Carreau et al, 2001;Kurosumi, Ishimura, Fujita, & Osawa, 1985;Nitta et al, 1993).…”

“…Cyp19a1a was found to be mainly expressed in the ovarian follicle cell layer and especially in granulosa cells of fish (Chaube, Rawat, & Joy, ; Dong & Willett, ; Goto‐Kazeto, Kight, Zohar, Place, & Trant, ; Kobayashi, Kajiura‐Kobayashi, & Nagahama, ; Wang et al, ). Cyp19a1a deficiency resulted in female‐to‐male sex reversal in zebrafish ( Danio rerio ) and tilapia ( Oreochromis niloticus ); by contrast, no reproductive defects were observed in the mutant males of these two species (Dranow et al, ; Lau, Zhang, Qin, & Ge, ; Yin et al, ; Zhang et al, ), suggesting a key role for Cyp19a1a in female fish sex determination, with little contribution to male reproductive function. Cyp19a1b was reported to be expressed in the testes of fish (Caulier et al, ; Chaube et al, ; Johnsen, Tveiten, Torgersen, & Andersen, ; Patel, Scheffler, Wang, & Willett, ; Tang, Hu, Hao, & Zhu, ; von Schalburg et al, ).…”

High-quality chromosome-level genomes of two tilapia species reveal their evolution of repeat sequences and sex chromosomes

“…This suggests that these genes participate in the SD process of both species, but may play a different role because of the turnover of their upstream SD genes. Among them, knockouts in ONJp of conserved teleost male-determining genes Amhy, Gsdf or Dmrt1 , or those of female-determining genes Foxl2 or Cyp19a1a all leads to sex reversal [27, 51, 52]. The temporal expression patterns of these genes are consistent with their known hierarchical positions in the SD pathway of ONJp : for example, Dmrt1 has robust male-specific expression and steady upregulation since 5dah, before its validated downstream target genes Gsdf [52], Sox9b [53] and Sox30 [54] reaching their peak expression levels specifically in males.…”

“…They will be very useful for anchoring the genomes of other cichlid species into chromosomes, and annotating their genes and conserved functional non-coding regulatory elements. More importantly, so far a total of 6 SD genes have been successfully knocked out in ONJp [25, 27, 51, 52, 75], with other well-established genetic resources and techniques like antibodies and transgenics, as well as the high-quality genome available now, ONJp becomes a promising model for testing the functions of identified genes responsible for the focal phenotypes (e.g., sex determination, body colors) in the future.…”

A Review of Genetic Advances Related to Sex Control and Manipulation in Tilapia

“…However, the fertility of these mutants remains to be tested. We also found that mutation of foxl2 or cyp19a1a resulted in female-to-male sex reversal (Zhang et al 2017). Crossing XX cyp19a1a−/− (♂) or XX foxl2−/− (♂) pseudomales with XX females may be a potential method to produce all XX populations.…”